U.S. patent number 9,168,266 [Application Number 14/352,096] was granted by the patent office on 2015-10-27 for hybrid diazeniumdiolated compounds, pharmaceutical compositions, and method of treating cancer.
This patent grant is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Services. The grantee listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Services, The United States of America, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Xinhua Ji, Larry K. Keefer, Vandana Kumari, Anna E. Maciag, Joseph E. Saavedra.
United States Patent |
9,168,266 |
Maciag , et al. |
October 27, 2015 |
Hybrid diazeniumdiolated compounds, pharmaceutical compositions,
and method of treating cancer
Abstract
Disclosed are hybrid compounds that release both nitric oxide
and a moiety that inhibits poly (ADP-ribose) polymerase (PARP),
e.g., a compound or a pharmaceutically acceptable salt thereof of
formula (I), wherein R.sup.1-4 and m-p are as described herein.
Also disclosed are pharmaceutical compositions and methods of use
including treating cancer and enhancing the chemotherapeutic
treatment of chemotherapeutic agents and high energy radiation.
##STR00001##
Inventors: |
Maciag; Anna E. (Frederick,
MD), Keefer; Larry K. (Bethesda, MD), Saavedra; Joseph
E. (Thurmont, MD), Ji; Xinhua (Frederick, MD),
Kumari; Vandana (Frederick, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Services |
Washington |
DC |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Services (Washington, DC)
|
Family
ID: |
48141619 |
Appl.
No.: |
14/352,096 |
Filed: |
October 18, 2012 |
PCT
Filed: |
October 18, 2012 |
PCT No.: |
PCT/US2012/060785 |
371(c)(1),(2),(4) Date: |
April 16, 2014 |
PCT
Pub. No.: |
WO2013/059433 |
PCT
Pub. Date: |
April 25, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20140315865 A1 |
Oct 23, 2014 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61549862 |
Oct 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/704 (20130101); C07D 237/32 (20130101); C07D
237/30 (20130101); A61P 35/00 (20180101); A61K
31/655 (20130101); C07C 291/02 (20130101); A61K
45/06 (20130101); A61K 31/69 (20130101); C07D
403/10 (20130101); C07D 295/28 (20130101); A61K
31/69 (20130101); A61K 2300/00 (20130101); A61K
31/704 (20130101); A61K 2300/00 (20130101) |
Current International
Class: |
C07D
403/10 (20060101); A61K 31/655 (20060101); A61K
45/06 (20060101); A61K 31/69 (20060101); C07D
295/28 (20060101); C07C 291/02 (20060101); C07D
237/32 (20060101); A61K 31/704 (20060101); C07D
237/30 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2780633 |
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May 2011 |
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CA |
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WO 2011/060215 |
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May 2011 |
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WO |
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Other References
Chakrapani et al., "Synthesis and in vitro anti-leukemic activity
of structural analogues of JS-K, an anti-cancer lead compound,"
Bioorg. & Med. Chem. Lett., (2008) 950-953, 18. cited by
applicant .
Chakrapani et al., "Synthesis, mechanistic studies, and
anti-proliferative activity of glutathione/glutathione
S-transferase-activated nitric oxide prodrugs," Bioorg. & Med.
Chem., (Sep. 30, 2008) 9764-9771, 16. cited by applicant .
International Preliminary Report on Patentability, Application No.
PCT/US2012/060785, dated Apr. 22, 2014. cited by applicant .
International Search Report, Application No. PCT/US2012/060785,
dated Jul. 1, 2013. cited by applicant .
Kiziltepe et al., "JS-K, a GST-activated nitric oxide generator,
induces DNA double-strand breaks, activates DNA damage response
pathways, and induces apoptosis in vitro and in vivo in human
multiple myeloma cells," Blood, (2007) 709-718, 110, 2. cited by
applicant .
Maciag et al., "The Nitric Oxide Prodrug JS-K Is Effective against
Non-small-Cell Lung Cancer Cells in vitro and in vivo: Involvement
of Reactive Oxygen Species," The Journal of Pharmacology and
Experimental Therapeutics, (Oct. 20, 2010) 313-320, 336, 2. cited
by applicant .
Maciag et al., "Nitric Oxide (NO) Releasing Poly ADP-ribose
Polymerase 1 (PARP-1) Inhibitors Targeted to Glutathione
S-Transferase P1-Overexpressing Cancer Cells," J. Med. Chem.,
(2014) 2292-2302, 57. cited by applicant .
Menear et al., "Novel alkoxybenzamide inhibitors of
poly(ADP-ribose) polymerase," Bioorg. Med. Chem. Lett., (2008)
3942-3945, 18. cited by applicant .
Menear et al.,
"4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-
fluorobenzyl]-2H-phthalazin-1-one: A Novel Bioavailable Inhibitor
of Poly(ADP-ribose) Polymerase-1," J. Med. Chem., (2008) 6581-6591,
51. cited by applicant .
Nandurdikar et al., "Synthesis and evaluation of piperazine and
homopiperazine analogues of JS-K, an anti-cancer lead compound,"
Bioorg. & Med. Chem. Lett., (2009) 2760-2762, 19. cited by
applicant .
Shami et al., "JS-K, a Glutathione/Glutathione
S-Transferase-activated Nitric Oxide Donor of the Diazeniumdiolate
Class with Potent Antineoplastic Activity," Mol. Cancer Ther.,
(2003) 409-417, 2. cited by applicant .
Shami et al., "Antitumor Activity of JS-K
|O.sup.2-(2,4-Dinitrophenyl)
1-[(4-Ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and
Related O.sup.2-Aryl Diazeniumdiolates in Vitro and in Vivo," J.
Med. Chem., (2006) 4356-4366, 49. cited by applicant .
Written Opinion of the International Searching Authority,
Application No. PCT/US2012/060785, dated Jul. 1, 2013. cited by
applicant.
|
Primary Examiner: Havlin; Robert
Attorney, Agent or Firm: Leydig, Voit & Mayer
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a U.S. National Phase of International
Patent Application No. PCT/US2012/060785, filed Oct. 18, 2012,
which claims the benefit of U.S. Provisional patent Application No.
61/549,862, filed Oct. 21, 2011, each of which is incorporated by
reference.
Claims
The invention claimed is:
1. A compound of formula (I) ##STR00021## wherein R.sup.1 is
selected from H, CN, NO.sub.2, NCS, SCN, F, Cl, Br, I, and
OCF.sub.3; R.sup.2 is H; R.sup.3 is selected from ##STR00022##
wherein R.sup.5 and R.sup.6 are each individually selected from H,
acyl, and C.sub.1-6 alkyl; R.sup.7 is selected from H, halo,
NO.sub.2, cyano, OH, alkoxy, mercapto, thioalkoxy, amino, C.sub.1-6
alkyl, C.sub.1-7 haloalkyl, heterocycloalkyl, and aryl; X is
selected from --CH.sub.2--, --CH.sub.2CH.sub.2--, --O--,
--OCH.sub.2--, --CH.sub.2O--, --NR.sup.8--, --CH.sub.2NR.sup.8--,
and --NR.sup.8CH.sub.2--; wherein R.sup.8 is selected from H and
C.sub.1-C.sub.6 alkyl; and q is 0 to 4; R.sup.4 is independently
selected from H, halo, OH, CN, NO.sub.2, sulfonato, formyl,
carboxy, mercapto, amido, amino, or a moiety selected from alkyl,
alkenyl, alkynyl, aryl alkyl, aryl, heterocycloalkyl, heteroaryl,
heteroaryl alkyl, hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy,
aryloxy, thioalkoxy, acyl, acyloxy, alkoxycarbonyl,
alkoxycarbonyloxy, alkylamino, and dialkylamino, and wherein each
moiety is optionally further substituted with one or more
substituents selected from alkyl, alkenyl, alkynyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and dialkylamino; m
is independently 0 to 5; n and o are independently 0 to 4; and p is
1; or a pharmaceutically acceptable salt thereof.
2. The compound or salt of claim 1, wherein R.sup.4 is H, halo, OH,
or an alkyl optionally substituted with one or more substituents
selected from alkyl, alkenyl, alkynyl, aryl alkyl, aryl,
heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino.
3. The compound or salt of claim 1, wherein the compound of formula
(I) is selected from compounds a-d: ##STR00023## ##STR00024##
4. A pharmaceutical composition comprising the compound of claim 1
or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier.
5. A method of treating non-small cell lung cancer in a patient
comprising administering to the patient an effective amount of the
compound of claim 1 or a pharmaceutically acceptable salt
thereof.
6. The method of claim 5, wherein the non-small cell lung cancer
has a reactive oxygen species (ROS) content greater than the ROS
content of a corresponding nonmalignant cell.
7. The method of claim 5, wherein the non-small cell lung cancer
has an 8-oxo-dG DNA glycosylase (OGG1) content less than the OGG1
content of a corresponding nonmalignant cell.
8. The method of claim 5, further comprising co-administering a
chemotherapeutic agent or with high energy radiation to the
patient.
9. A method of enhancing chemotherapeutic treatment of a cancer
patient with a chemotherapeutic agent that produces reactive oxygen
species (ROS) in a cancer cell or radiation treatment of cancer,
the method comprising administering to the patient an effective
amount of the compound of claim 1 or a pharmaceutically acceptable
salt thereof, wherein the patient has non-small cell lung
cancer.
10. The compound or salt of claim 1, wherein R.sup.1 is H or
NO.sub.2.
11. The compound or salt of claim 1, wherein R.sup.3 is
##STR00025##
12. The compound or salt of claim 11, wherein R.sup.5 is H and
C.sub.1-6 alkyl.
13. The compound or salt of claim 11, wherein q is 0.
14. The compound or salt of claim 11, wherein X is --CH.sub.2-- or
--CH.sub.2CH.sub.2--.
15. The compound or salt of claim 1, wherein R.sup.3 is
##STR00026##
16. The compound or salt of claim 15, wherein R.sup.5 is H and
C.sub.1-6 alkyl.
17. The compound or salt of claim 15, wherein q is 0.
18. The compound or salt of claim 15, wherein X is --O--,
--OCH.sub.2--, or --CH.sub.2O--.
19. The compound or salt of claim 1, wherein R.sup.4 is H or
halo.
20. The compound or salt of claim 1, wherein the compound of
formula (I) is compound a: ##STR00027##
21. The compound or salt of claim 1, wherein the compound of
formula (I) is compound b: ##STR00028##
22. The compound or salt of claim 1, wherein the compound of
formula (I) is compound c: ##STR00029##
23. The compound or salt of claim 1, wherein the compound of
formula (I) is compound d: ##STR00030##
Description
BACKGROUND OF THE INVENTION
Poly(ADP-ribose) polymerase (PARP) is an attractive antitumor
target because of its vital role in DNA repair. Many anti-cancer
therapies, including alkylating agents and radiation, produce DNA
strand breaks and PARP is an essential player in the repair of this
type of DNA damage. PARP inhibitors have emerged as a promising
therapeutic class of compounds, and numerous PARP inhibitors have
advanced into clinical trials.
The homologous recombination (HR) DNA repair pathway is critical
for the repair of DNA double-strand breaks. HR deficiency leads to
a dependency on error-prone DNA repair mechanisms, with consequent
genomic instability and oncogenesis. Tumor-specific HR defects may
be exploited through a synthetic lethal approach for the
application of anticancer therapeutics, including PARP inhibitors.
The demonstration of single-agent antitumor activity of PARP
inhibitors in cancers with deficiencies in breast cancer
susceptibility BRCA1 and BRCA2 provides strong evidence for the
clinical application of this approach. Mutations in the BRCA1/2
genes are associated with HR-mediated double strand break repair
defects, and inhibition of the base-excision repair-mediated single
strand break repair via PARP inhibition results in synthetic
lethality. For example, olaparib (AZD-2281/KU-0059436, Astra
Zeneca) is a phthalazinone PARP inhibitor that is in phase II
clinical trials as an oral single agent for the treatment of
BRCA-deficient breast and ovarian cancers (Vasiliou at al., Drugs
Future, 34: 101-105 (2009)).
Nitric oxide (NO) is a signaling molecule, a toxicant, and an
antioxidant under various conditions, with a broad spectrum of
actions in physiological and pathological processes.
Diazeniumdiolate-based nitric oxide-releasing prodrugs are a
growing class of promising NO-based cancer therapeutics.
O.sup.2-(2,4-Dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1--
ium-1,2-diolate (JS-K) has proven effective against leukemia,
multiple myeloma, prostate, liver, and non-small cell lung cancer
(NSCLC) cancer cell lines in vitro and in vivo (see, e.g., Shami et
al., Mol. Cancer Ther., 2: 409-417 (2003); Shami et al., J. Med.
Chem., 49: 4356-4366 (2006); Kiziltepe et al., Blood, 110: 709-718
(2007); and Maciag et al., J. Pharmacol. Exp. Ther., 336: 313-320
(2011)).
Thus, even though current therapies exist, there is an unmet need
for agents suitable for treating cancers, particularly agents that
can both damage DNA and inhibit DNA repair in cancer cells.
BRIEF SUMMARY OF THE INVENTION
The invention provides novel hybrid compounds which act as dual
prodrugs comprising a functional portion of a PARP inhibitor and a
diazeniumdiolate moiety N.sub.2O.sub.2.sup.-. Accordingly, the
compounds of the invention release both a PARP-inhibiting moiety
and nitric oxide under physiological conditions.
The invention provides a hybrid diazeniumdiolated compound of
formula (I)
##STR00002## in which
R.sup.1 is H or a moiety independently selected from N.sub.3, CN,
NO.sub.2, CHO, NCS, SCN, F, Cl, Br, I, OCF.sub.3,
O--N.dbd.N(O)NR'.sub.2, SO.sub.3H, B(OH).sub.2, PO(OH).sub.2,
PO(OH)(OR'), PO(OR').sub.2, SO.sub.2NHOH, SO.sub.2NH.sub.2,
CONH.sub.2, CONHOH, SR', SOR', SO.sub.2R', SO.sub.2NHR',
SO.sub.2N(R')R', SO.sub.2NHCON(R')R', COOR', COR', CONHR',
CON(R')R', CONHSO.sub.2N(R')R', NHCOR', N(R')COR', NHSO.sub.2R',
N(R')SO.sub.2R', NH.sub.2R'.sup.+M.sup.-, NHR'.sub.2.sup.+M.sup.-,
and NR'.sub.3.sup.+M.sup.-, wherein each R' is the same or
different and is selected from H, C.sub.1-C.sub.6 alkyl, and
CY.sub.3, wherein Y is F, Cl, or Br; M.sup.- is a counterion;
wherein the C.sub.1-C.sub.6 alkyl is optionally substituted with
one or more substituents selected from halo, OH, alkoxy, CN, amino,
and NO.sub.2;
R.sup.2 is independently selected from H, CN, formyl, carboxy,
amido, and a moiety selected from alkyl, alkenyl, alkynyl, aryl
alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, and alkoxycarbonyloxy, and wherein
each moiety is optionally further substituted with one or more
substituents selected from alkyl, alkenyl, alkynyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino;
R.sup.3 comprises
a moiety selected from aryl, heteroaryl, and heterocycloalkyl, each
of which is optionally substituted with at least one substituent
selected from NHR.sup.5CO, OR.sup.6, carboxy, carboxyalkyl, amido,
alkyl, NO.sub.2, halo, mercapto, thioalkoxy, cyano, alkoxy,
C.sub.2-7 haloalkyl, heterocycloalkyl, and aryl, wherein R.sup.5
and R.sup.6 are each individually selected from H, acyl, and
C.sub.1-6 alkyl, and
a linker, wherein the moiety is attached to phenyl ring A through
the linker;
R.sup.4 is independently selected from H, halo, OH, CN, NO.sub.2,
sulfonato, formyl, carboxy, mercapto, amido, amino, or a moiety
selected from alkyl, alkenyl, alkynyl, aryl alkyl, aryl,
heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and dialkylamino,
and wherein each moiety is optionally further substituted with one
or more substituents selected from alkyl, alkenyl, alkynyl, aryl
alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino;
m is independently 0 to 5; n and o are independently 0 to 4;
and
p is 1 or 2;
or a pharmaceutically acceptable salt thereof.
The invention further provides a hybrid diazeniumdiolated compound
of formula (II)
##STR00003## wherein
R.sup.1' and R.sup.2' are each independently selected from alkyl,
alkyl substituted with alkoxy, acyloxy, OH, halo, or benzyl,
alkenyl, and alkenyl substituted with alkoxy, acyloxy, OH, halo, or
benzyl, or R.sup.1' and R.sup.2', together with the nitrogen atom
to which they are bonded, form a heterocyclyl, e.g.,
heterocycloalkyl, having at least one or more heteroatoms selected
from O, S and N;
R.sup.3' is independently selected from H, CN, NO.sub.2, sulfonato,
formyl, carboxy, mercapto, amido, amino, alkyl, alkenyl, alkynyl,
aryl alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino, wherein each of said alkyl, aryl alkyl, aryl,
heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, alkylamino, and
dialkylamino is optionally substituted with one or more
substituents selected from alkyl, alkenyl, alkynyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, OH,
hydroxyalkyl, halo, haloalkyl, aminoalkyl, alkoxy, aryloxy,
thioalkoxy, acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy,
alkylamino, and dialkylamino;
m is independently 0 to 4;
Y is a linker selected from --NR.sup.9'--,
##STR00004## --S--, and --O--, wherein R.sup.9' is selected from H
and C.sub.1-C.sub.6 alkyl, and s is 0 to 4;
R.sup.4' is a moiety selected from
##STR00005## wherein
R.sup.5' and R.sup.6' are each independently selected from H, acyl,
and C.sub.1-6 alkyl;
R.sup.7' is each independently selected from H, halo, NO.sub.2, CN,
OH, alkoxy, mercapto, thioalkoxy, amino, C.sub.1-6 alkyl, C.sub.2-7
haloalkyl, heterocycloalkyl, and aryl;
R.sup.10' is each independently selected from H, halo, OH, CN,
NO.sub.2, sulfonato, formyl, carboxy, mercapto, amido, amino, or an
optionally substituted moiety selected from alkyl, alkenyl,
alkynyl, aryl alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl
alkyl, hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy,
thioalkoxy, acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy,
alkylamino, and dialkylamino;
X' is a linker selected from --(CH.sub.2).sub.r--, --O--, --C(O)--,
--O(CH.sub.2).sub.r--, --(CH.sub.2).sub.rO--, --S--,
--S(CH.sub.2).sub.r--, --(CH.sub.2).sub.rS--, --NR.sup.8'--,
--(CH.sub.2).sub.rNR.sup.8'--, --NR.sup.8'(CH.sub.2).sub.r--,
--NR.sup.8'C(O)NR.sup.8'--, and --C(O)NR.sup.8'C(O)--; wherein r is
1 to 5; and R.sup.8' is selected from H and C.sub.1-C.sub.6 alkyl;
and
q and r are independently 0 to 4;
or a pharmaceutically acceptable salt thereof.
The invention also provides pharmaceutical compositions and methods
of treating cancer by the use of the above compounds. For example,
in one aspect, the method is applicable to treating cancers wherein
the cancer cell has an elevated ROS content and/or decreased levels
of OGG1. The invention further provides a method for enhancing the
chemotherapeutic treatment of cancer and/or radiation treatment of
cancer.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIG. 1 depicts a schematic illustrating a compound of formula (I)
and a proposed mechanism of its decomposition in the presence of
glutathione (GSH).
FIG. 2 depicts a reaction scheme illustrating the synthesis of
compounds in accordance with an embodiment of the invention:
JS-59-183 and JS-60-178.
FIG. 3 depicts a reaction scheme illustrating the synthesis of a
compound in accordance with an embodiment of the invention:
JS-59-13.
FIG. 4A depicts the intracellular NO release from JS-59-183 and
JS-60-178 in H1703 lung adenocarcinoma cells measured as DAF
fluorescence.
FIG. 4B depicts illustrating the intracellular ROS/RNS level of
JS-59-183 and JS-60-178 measured as DCF fluorescence. (n=8);
***P<0.0001, by paired t test, compared with cells treated with
DMSO only.
FIG. 5 depicts inhibition curves of PARP enzyme by JS-60-178
(.circle-solid.) and JS-59-183 (), compared with Olaparib
(.box-solid.). The inhibitors were tested in concentration range of
0.1 nM-10 .mu.M.
FIG. 6 is a series of graphs depicting the toxicity (expressed as
IC.sub.50 and TGI) of JS-60-178 correlated with levels of
endogenous ROS/RNS (measured as DCF fluorescence), with levels of
DNA repair protein OGG1, and with levels of ROS/RNS scavenger
peroxiredoxin 1 (PRX1).
DETAILED DESCRIPTION OF THE INVENTION
It is envisioned that the hybrid compounds of the invention can
deliver DNA-damaging NO and a PARP inhibitor simultaneously to a
cancer cell. FIG. 1 illustrates an example of a prodrug of the
invention and a proposed mechanism of NO/PARP inhibitor release. It
is believed that the inventive prodrugs are activated by
glutathione/glutathione S-transferase P1 (GSH/GSTP1), a phase II
detoxifying enzyme that is frequently overexpressed in cancer
cells, and release cytotoxic NO and a PARP inhibitor in the target
cancer cell. Preferably the target cell is a cancer cell that is
high in GSH/GSTP1. Therefore, the compounds of the present
invention allow a concurrent release of cytotoxic components upon
metabolic activation in the cancer cell, which should have
advantages over delivering two independent conventional therapeutic
molecules.
In accordance with an embodiment, the invention provides a hybrid
compound of formula (I)
##STR00006## wherein
R.sup.1 is H or a moiety independently selected from N.sub.3, CN,
NO.sub.2, CHO, NCS, SCN, F, Cl, Br, I, OCF.sub.3,
O--N.dbd.N(O)NR'.sub.2, SO.sub.3H, B(OH).sub.2, PO(OH).sub.2,
PO(OH)(OR'), PO(OR').sub.2, SO.sub.2NHOH, SO.sub.2NH.sub.2,
CONH.sub.2, CONHOH, SR', SOR', SO.sub.2R', SO.sub.2NHR',
SO.sub.2N(R')R', SO.sub.2NHCON(R')R', COOR', COR', CONHR',
CON(R')R', CONHSO.sub.2N(R')R', NHCOR', N(R')COR', NHSO.sub.2R',
N(R')SO.sub.2R', NH.sub.2R'.sup.+M.sup.-, NHR'.sub.2.sup.+M.sup.-,
and NR'.sub.3.sup.+M.sup.-, wherein each R' is the same or
different and is selected from H, C.sub.1-C.sub.6 alkyl, and
CY.sub.3, wherein Y is F, Cl, or Br; M.sup.- is a counterion;
wherein the C.sub.1-C.sub.6 alkyl is optionally substituted with
one or more substituents selected from halo, OH, alkoxy, CN, amino,
and NO.sub.2;
R.sup.2 is independently selected from H, CN, formyl, carboxy,
amido, and a moiety selected from alkyl, alkenyl, alkynyl, aryl
alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, and alkoxycarbonyloxy, and wherein
each moiety is optionally further substituted with one or more
substituents selected from alkyl, alkenyl, alkynyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino;
R.sup.3 comprises
a moiety selected from aryl, heteroaryl, and heterocycloalkyl, each
of which is optionally substituted with at least one substituent
selected from NHR.sup.5CO, OR.sup.6, carboxy, carboxyalkyl, amido,
alkyl, NO.sub.2, halo, mercapto, thioalkoxy, cyano, alkoxy,
C.sub.2-7 haloalkyl, heterocycloalkyl, and aryl, wherein R.sup.5
and R.sup.6 are each individually selected from H, acyl, and
C.sub.1-6 alkyl, and
a linker, wherein the moiety is attached to phenyl ring A through
the linker;
R.sup.4 is independently selected from H, halo, OH, CN, NO.sub.2,
sulfonato, formyl, carboxy, mercapto, amido, amino, or a moiety
selected from alkyl, alkenyl, alkynyl, aryl alkyl, aryl,
heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and dialkylamino,
and wherein each moiety is optionally further substituted with one
or more substituents selected from alkyl, alkenyl, alkynyl, aryl
alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino;
m is independently 0 to 5; n and o are independently 0 to 4;
and
p is 1 or 2;
or a pharmaceutically acceptable salt thereof.
In certain embodiments, R.sup.1 is H or a moiety independently
selected from N.sub.3, CN, NO.sub.2, CHO, NCS, SCN, F, Cl, Br, I,
OCF.sub.3, SO.sub.3H, B(OH).sub.2, PO(OH).sub.2, PO(OH)(OR'),
PO(OR').sub.2, SO.sub.2NHOH, SO.sub.2NH.sub.2, CONH.sub.2, CONHOH,
SR', SOR', SO.sub.2R', SO.sub.2NHR', SO.sub.2N(R')R',
SO.sub.2NHCON(R')R', COOR', COR', CONHR', CON(R)R',
CONHSO.sub.2N(R')R', NHCOR', N(R')COR', NHSO.sub.2R',
N(R)SO.sub.2R', NH.sub.2R'.sup.+M.sup.-, NHR'.sub.2.sup.+M.sup.-,
and NR'.sub.3.sup.+M.sup.-, wherein each R' is the same or
different and is selected from H, C.sub.1-C.sub.6 alkyl, and
CY.sub.3, wherein Y is F, Cl, or Br; M.sup.- is a counterion (e.g.,
hydride, fluoride, chloride, bromide, iodide, nitride, phosphate,
hydrogen phosphate, dihydrogen phosphate, sulfate, hydrogen
suldate, nitrate, nitrite, chlorate, chlorite, hypochlorite,
hypobromite, carbonate, hydrogen carbonate, acetate, formate,
hydroxide, cyanide, and thiocyanate); wherein the C.sub.1-C.sub.6
alkyl is optionally substituted with one or more substituents
selected from halo, OH, alkoxy, CN, amino, and NO.sub.2. In other
embodiments, R.sup.1 is selected from H, CN, NO.sub.2, NCS, SCN, F,
Cl, Br, I, and OCF.sub.3. Preferably, R.sup.1 is CN or NO.sub.2.
More preferably, R.sup.1 is NO.sub.2.
In any of the foregoing embodiments or other embodiments, R.sup.2
is H, CN, NO.sub.2, carboxy, or an optionally substituted moiety
selected from alkyl, aryl alkyl, aryl, heterocycloalkyl,
heteroaryl, heteroaryl alkyl, hydroxyalkyl, haloalkyl, aminoalkyl,
alkoxy, aryloxy, alkylamino, and dialkylamino. Preferably, R.sup.2
is H or an optionally substituted alkyl. More preferably, R.sup.2
is H.
In any of the foregoing embodiments or other embodiments, R.sup.3
comprises a moiety selected from aryl and heteroaryl, each of which
is optionally substituted with at least one substituent selected
from NHR.sup.5CO, OR.sup.6, alkyl, NO.sub.2, halo, CN, alkoxy, and
C.sub.1-7 haloalkyl, wherein R.sup.5 and R.sup.6 are each
individually selected from H, acetyl, and C.sub.1-6 alkyl, and a
linker, wherein the moiety is attached to the phenyl ring through
the linker. In an aspect, the linker of R.sup.3 is selected from
--(CH.sub.2).sub.r--, --O--, --C(O)--, --O(CH.sub.2).sub.r--,
--(CH.sub.2).sub.rO--, --S--, --S(CH.sub.2).sub.r,
--(CH.sub.2).sub.rS--, --NR.sup.8--, --(CH.sub.2).sub.rNR.sup.8-,
NR.sup.8(CH.sub.2).sub.r--, --NR.sup.8C(O)NR.sup.8--, and
--C(O)NR.sup.8C(O)--; wherein r is 1 to 5; and R.sup.8 is selected
from H and C.sub.1-C.sub.6 alkyl. Preferably, R.sup.3 is a moiety
selected from
##STR00007## wherein
R.sup.5 and R.sup.6 are each individually selected from H, acetyl,
and C.sub.1-6 alkyl;
R.sup.7 is selected from H, halo, NO.sub.2, CN, OH, alkoxy,
mercapto, thioalkoxy, amino, C.sub.1-6 alkyl, C.sub.1-7 haloalkyl,
heterocycloalkyl, and aryl;
X is a linker selected from --CH.sub.2--, --CH.sub.2CH.sub.2--,
--O--, --OCH.sub.2--, --CH.sub.2O--, --NR.sup.8--,
--CH.sub.2NR.sup.8--, and --NR.sup.8CH.sub.2--; wherein R.sup.8 is
selected from H and C.sub.1-C.sub.6 alkyl; and
q is 0 to 4.
In a preferred embodiment, R.sup.3 is
##STR00008##
Particularly preferred are compounds in which R.sup.3 is
##STR00009##
In any of the foregoing embodiments or other embodiments, R.sup.3
can be present in formula (I) at any suitable position on the
phenyl ring (e.g., the 1-, 2-, 3-, 4- or 5-position). Similarly,
the substituent(s) R.sup.4 are attached to the phenyl ring at any
suitable position (e.g., the 1-, 2-, 3-, 4- or 5-position). In
certain embodiments, R.sup.3 is present in formula (I) at the
5-position on the phenyl ring. Additionally, R.sup.4 preferably is
attached to the phenyl ring at 2- and/or 4-positions.
In any of the foregoing embodiments or other embodiments, R.sup.7
is H, OH, halo, or C.sub.1-6 alkyl. Preferably, R.sup.7 is H or
halo.
In any of the foregoing embodiments or other embodiments, R.sup.4
is H, OH, halo, or C.sub.1-6 alkyl. Preferably, R.sup.4 is H or
halo.
In any of the embodiments, m is independently 0 to 4. In certain
embodiments, m is 1, 2, 3, or 4.
In any of the foregoing embodiments, m preferably is 1 or 2 and/or
n preferably is 0 or 1 and/or p preferably is 1 and/or o preferably
is 0, 1, or 2.
In accordance with another embodiment, the invention provides a
hybrid compound of formula (II)
##STR00010## wherein
R.sup.1' and R.sup.2' are each independently selected from alkyl,
alkyl substituted with alkoxy, acyloxy, OH, halo, or benzyl,
alkenyl, and alkenyl substituted with alkoxy, acyloxy, OH, halo, or
benzyl, or R.sup.1' and R.sup.2', together with the nitrogen atom
to which they are bonded, form a heterocyclyl, e.g.,
heterocycloalkyl, having at least one or more heteroatoms selected
from O, S and N;
R.sup.3' is independently selected from H, CN, NO.sub.2, sulfonato,
formyl, carboxy, mercapto, amido, amino, alkyl, alkenyl, alkynyl,
aryl alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino, wherein each of said alkyl, aryl alkyl, aryl,
heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, alkylamino, and
dialkylamino is optionally substituted with one or more
substituents selected from alkyl, alkenyl, alkynyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, OH,
hydroxyalkyl, halo, haloalkyl, aminoalkyl, alkoxy, aryloxy,
thioalkoxy, acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy,
alkylamino, and dialkylamino;
m is 0 to 4;
Y is a linker selected from the group consisting of
--NR.sup.9'--,
##STR00011## --S--, and --O--, wherein R.sup.9' is selected from
the group consisting of H and C.sub.1-C.sub.6 alkyl, and s is 0 to
4;
R.sup.4' is a moiety selected from
##STR00012## wherein
R.sup.5' and R.sup.6' are each independently selected from H, acyl,
and C.sub.1-6 alkyl;
R.sup.7' is each independently selected from H, halo, NO.sub.2, CN,
OH, alkoxy, mercapto, thioalkoxy, amino, C.sub.1-6 alkyl, C.sub.2-7
haloalkyl, heterocycloalkyl, and aryl;
R.sup.10' is each independently selected from H, halo, OH, CN,
NO.sub.2, sulfonato, formyl, carboxy, mercapto, amido, amino, or an
optionally substituted moiety selected from alkyl, alkenyl,
alkynyl, aryl alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl
alkyl, hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy,
thioalkoxy, acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy,
alkylamino, and dialkylamino;
X' is a linker selected from (CH.sub.2).sub.r--, --O--, --C(O)--,
--O(CH.sub.2).sub.r--, --(CH.sub.2).sub.rO--, --S--,
--S(CH.sub.2).sub.r--, --(CH.sub.2).sub.rS--, --NR.sup.8'--,
--(CH.sub.2).sub.rNR.sup.8'--, NR.sup.8'(CH.sub.2).sub.r,
--NR.sup.8'C(O)NR.sup.8'--, and --C(O)NR.sup.8'C(O)--; wherein r is
1 to 5; and R.sup.8' is selected from H and C.sub.1-C.sub.6 alkyl;
and
q and r are independently 0 to 4;
or a pharmaceutically acceptable salt thereof.
In certain embodiments, R.sup.1' and R.sup.2' are each individually
selected from alkyl and alkyl substituted by alkoxy, acyloxy, OH,
halo, or benzyl. In other embodiments, R.sup.1' and R.sup.2' join
together with the nitrogen atom to which they are bonded to form a
piperidinyl, pyrrolidinyl, piperazinyl, or morpholinyl.
In any of the foregoing embodiments or other embodiments, R.sup.3'
is independently selected from H, CN, NO.sub.2, sulfonato, formyl,
carboxy, mercapto, amino, amino, alkyl, alkenyl, alkynyl, aryl
alkyl, aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl,
hydroxyalkyl, haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy,
acyl, acyloxy, alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and
dialkylamino, wherein each of said alkyl, aryl alkyl, aryl,
heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, alkylamino, and
dialkylamino is optionally substituted with one or more
substituents selected from alkyl, alkenyl, alkynyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, thioalkoxy, acyl, acyloxy,
alkoxycarbonyl, alkoxycarbonyloxy, alkylamino, and dialkylamino. In
certain embodiments, R.sup.3' is H, CN, NO.sub.2, carboxy, or an
optionally substituted moiety selected from alkyl, aryl alkyl,
aryl, heterocycloalkyl, heteroaryl, heteroaryl alkyl, hydroxyalkyl,
haloalkyl, aminoalkyl, alkoxy, aryloxy, alkylamino, and
dialkylamino. Preferably, R.sup.3' is NO.sub.2.
In any of the foregoing embodiments or other embodiments, in is 0,
1, 2, or 3. Preferably, m is 2.
In any of the foregoing embodiments or other embodiments, Y is a
linker selected from the group consisting of --NR.sup.9'--, --S--,
and --O--, wherein R.sup.9' is selected from the group consisting
of H and C.sub.1-C.sub.6 alkyl. In certain embodiments, Y is
--NR.sup.9'- or --O--, wherein R.sup.9' is selected from H and
C.sub.1-C.sub.4 alkyl. When Y
##STR00013## is either the amino or oxy terminus can be bonded to
the carbonyl moiety of the compound of formula (II). Preferably,
the oxy terminus is connected to the carbonyl. Furthermore, when Y
is
##STR00014## the --NR.sup.9'-moiety can be at any suitable position
on the phenyl ring (e.g., the 2-, 3-, 4-, 5-, or 6-position). In
certain embodiments, the --NR.sup.9'-moiety is attached to the
phenyl ring at the 3- or 4-position.
In any of the foregoing embodiments or other embodiments, R.sup.4'
is attached to the core structure of formula (II) at any suitable
position on the phenyl ring (e.g., the 1-, 2-, 3-, 4- or
5-position). Similarly, the substituent(s) R.sup.10' are attached
to the phenyl ring at any suitable position (e.g., the 1-, 2-, 3-,
4- or 5-position). The aryl, heteroaryl, or heterocycloalkyl
moiety, connected via linker X', is attached at any suitable
position to the phenyl ring (e.g., the 1-, 2-, 3-, 4- or
5-position). In certain embodiments, R.sup.4' is attached to the
core structure of formula (II) at the 1-position on the phenyl
ring. Additionally, R.sup.10' preferably is attached at the 2-
and/or 4-positions, and the aryl, heteroaryl, or heterocycloalkyl
moiety connected via linker X' is attached at the 5-position on the
phenyl ring. Preferably, R.sup.4' is
##STR00015##
In any of the foregoing embodiments or other embodiments, R.sup.7'
is H, OH, halo, or C.sub.1-6 alkyl. Preferably, R.sup.7' is H or
halo (e.g., F, Cl, Br). In any of the foregoing embodiments or
other embodiments, R.sup.10' is H, OH, halo, or C.sub.1-6 alkyl.
Preferably, R.sup.10' is H or halo (e.g., F, Cl, Br). In any of the
foregoing embodiments, r preferably is 0, 1, or 2.
Some groups in the definitions of the substituents in formulae (I)
and (II), e.g., alkyl, alkenyl, aryl, or heterocyclyl, are
optionally substituted with one or more moieties (e.g., 1 to 5, 1
to 4, 1 to 3, 1 or 2). Suitable substituents are selected from
--[N(NO)O], halo, OH, alkylthio, arylthio, alkoxy, aryloxy,
carboxy, carboxyalkyl, alkylcarboxy, amino, alkylamino,
dialkylamino, nitroso, CN, sulfonato, mercapto, NO.sub.2, oxo
(.dbd.O), alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl alkyl,
aryl, benzylcarbonyl, phenylcarbonyl, phosphono, and phosphato.
Preferred substituents are selected from halo, OH, alkoxy, amino,
alkylamino, dialkylamino, CN, NO.sub.2, alkyl, alkenyl, cycloalkyl,
heterocycloalkyl, aryl alkyl, and aryl.
Specific examples of the compound of formula (I) include compounds
a-d:
##STR00016## ##STR00017##
Specific examples of the compound of formula (II) include compounds
e-i:
##STR00018## ##STR00019##
In any of the embodiments above, the term "alkyl" implies a
straight-chain or branched alkyl substituent containing from, for
example, about 1 to about 12 carbon atoms, preferably from about 1
to about 8 carbon atoms, more preferably from about 1 to about 6
carbon atoms. In accordance with an embodiment, the alkyl group is
preferably a C.sub.1-C.sub.3 alkyl. Examples of alkyl group include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, n-pentyl, isopentyl, n-hexyl, and the like. This
definition also applies wherever "alkyl" occurs as part of a group,
such as, e.g., in hydroxyalkyl, haloalkyl including monohalo alkyl,
dihalo alkyl, and trihalo alkyl, aminoalkyl, alkylamino,
dialkylamino, etc.
In any of the embodiments above, the term "alkenyl," as used
herein, means a linear alkenyl substituent containing from, for
example, about 2 to about 12 carbon atoms (branched alkenyls are
about 3 to about 12 carbons atoms), preferably from about 2 to
about 8 carbon atoms (branched alkenyls are preferably from about 3
to about 8 carbon atoms), more preferably from about 3 to about 6
carbon atoms. In accordance with an embodiment, the alkenyl group
is preferably a C.sub.2-C.sub.4 alkenyl. Examples of alkenyl group
include ethenyl, allyl, 2-propenyl, 1-butenyl, 2-butenyl,
1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, and the like.
In any of the embodiments above, the term "alkynyl," as used
herein, means a linear alkynyl substituent containing at least one
carbon-carbon triple bond and from, for example, about 2 to about
12 carbon atoms (branched alkynyls are about 4 to about 12 carbons
atoms), preferably from about 2 to about 8 carbon atoms (branched
alkynyls are preferably from about 4 to about 8 carbon atoms), more
preferably from about 3 to about 6 carbon atoms. Examples of such
substituents include propynyl, propargyl, n-butynyl, pentynyl,
isopentynyl, hexynyl, octynyl, dodecynyl, and the like.
In any of the embodiments above, the terms "hydroxy" and "thiol or
mercapto" refer to the groups --OH and --SH, respectively.
In any of the embodiments above, the terms "alkoxy" and
"thioalkoxy" embrace linear or branched alkyl groups that are
attached to a divalent oxygen or sulfur, respectively. The alkyl
group is the same as described herein. Examples of alkoxy group
include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,
sec-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy,
n-hexoxy, and the like. The term "aryloxy" refers to substituents
that have an aryl group attached to divalent oxygen. The aryl group
is the same as described herein. Examples of such substituents
include phenoxy.
In any of the embodiments above, the term "halo" refers to a
halogen selected from fluorine, chlorine, bromine, and iodine,
preferably fluorine, chlorine, or bromine.
In any of the embodiments above, the term "aryl" refers to a mono,
bi, or tricyclic carbocyclic ring system having one, two, or three
aromatic rings, for example, phenyl, naphthyl, anthracenyl, or
biphenyl. The term "aryl" refers to an unsubstituted or substituted
aromatic carbocyclic moiety, as commonly understood in the art, and
includes monocyclic and polycyclic aromatics such as, for example,
phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, and the like. An
aryl moiety generally contains from, for example, 6 to 30 carbon
atoms, preferably from 6 to 18 carbon atoms, more preferably from 6
to 14 carbon atoms and most preferably from 6 to 10 carbon atoms.
It is understood that the term aryl includes carbocyclic moieties
that are planar and comprise 4n+2.pi. electrons, according to
Huckel's Rule, wherein n=1, 2, or 3.
In any of the embodiments above, the term "heteroaryl" refers to
aromatic 5 or 6 membered monocyclic groups, 9 or 10 membered
bicyclic groups, and 11 to 14 membered tricyclic groups which have
at least one heteroatom (O, S or N) in at least one of the rings.
Each ring of the heteroaryl group containing a heteroatom can
contain one or two oxygen or sulfur atoms and/or from one to four
nitrogen atoms provided that the total number of heteroatoms in
each ring is four or less and each ring has at least one carbon
atom. The fused rings completing the bicyclic and tricyclic groups
may contain only carbon atoms and may be saturated, partially
saturated, or unsaturated. The nitrogen and sulfur atoms may
optionally be oxidized, and the nitrogen atoms may optionally be
quaternized. Heteroaryl groups which are bicyclic or tricyclic must
include at least one fully aromatic ring but the other fused ring
or rings may be aromatic or non-aromatic. The heteroaryl group may
be attached at any available nitrogen or carbon atom of any ring.
Illustrative examples of heteroaryl groups are pyridinyl,
pyridazinyl, pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl,
imidazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl, tetrazolyl,
furyl, pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and
oxadiazolyl.
In any of the embodiments above, the term "heterocyclyl" includes
heterocycloalkyl and heteroaryl groups.
The term "heterocycloalkyl" means a stable, saturated, or partially
unsaturated monocyclic, bicyclic, and spiro ring system containing
3 to 7 ring members of carbon atoms and other atoms selected from
nitrogen, sulfur, and/or oxygen. Preferably, a heterocycloalkyl is
a 5, 6, or 7-membered monocyclic ring and contains one, two, or
three heteroatoms selected from nitrogen, oxygen, and/or sulfur.
The heterocycloalkyl may be attached to the parent structure
through a carbon atom or through any heteroatom of the
heterocycloalkyl that results in a stable structure. Examples of
such heterocycloalkyl rings are isoxazolyl, thiazolinyl,
imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl,
pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl.
In any of the embodiments above, the term "aryl alkyl" as utilized
herein means alkyl as defined herein, wherein at least one hydrogen
atom is replaced with an aryl substituent as defined herein. Aryl
alkyls include, for example, benzyl, phenethyl, and groups of the
formula:
##STR00020##
In any of the embodiments above, the term "alkylamino" refers to a
secondary amine substituent with one hydrogen and one alkyl group
directly attached to a trivalent nitrogen atom. The term
"dialkylamino" refers to a tertiary amine substituent with two of
the same or different alkyl groups directly attached to a trivalent
nitrogen atom. The alkyl group is the same as described herein.
In any of the embodiments above, the term "carboxy" refers to the
group --C(O)OH. The term "carboxyalkyl" refers to the group
--RC(O)OH that is connected to the compound through the alkyl R
group. The term "alkoxycarbonyl" refers to the group --C(O)OR, in
which R is an alkyl group as described herein. The term
"alkoxycarbonyloxy" refers to the group --OC(O)OR, in which R is an
alkyl group as described herein. The term "formyl" refers to the
group --C(O)H. The term "acyl" refers to the group --C(O)R and the
teen "acyloxy" refers to the group --OC(O)R, in which R is an alkyl
group as described herein.
In any of the embodiments above, the term "amido" refers to the
group --C(O)NH.sub.2.
In any of the embodiments above, the term "sulfonato" refers to the
group --SO.sub.3.
In any of the embodiments above, the alkyl, alkoxy, and alkylamino
groups can be linear or branched. When an aryl group is substituted
with a substituent, e.g., halo, amino, alkyl, OH, alkoxy, and
others, the aromatic ring hydrogen is replaced with the substituent
and this can take place in any of the available hydrogens, e.g., 2,
3, 4, 5, and/or 6-position wherein the 1-position is the point of
attachment of the aryl group in the compound of the present
invention.
In any of the embodiments above, whenever a range of the number of
atoms in a structure is indicated (e.g., a C.sub.1-12, C.sub.1-8,
C.sub.1-6, or C.sub.1-4 alkyl, alkylamino, etc.), it is
specifically contemplated that any sub-range or individual number
of carbon atoms falling within the indicated range also can be
used. Thus, for instance, the recitation of a range of 1-8 carbon
atoms (e.g., C.sub.1-C.sub.8), 1-6 carbon atoms (e.g.,
C.sub.1-C.sub.6), 1-4 carbon atoms (e.g., C.sub.1-C.sub.4), 1-3
carbon atoms (e.g., C.sub.1-C.sub.3), or 2-8 carbon atoms (e.g.,
C.sub.2-C.sub.8) as used with respect to any chemical group (e.g.,
alkyl, alkylamino, etc.) referenced herein encompasses and
specifically describes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12
carbon atoms, as appropriate, as well as any sub-range thereof
(e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5
carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms,
1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms, 1-12 carbon
atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6
carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms,
2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms, 3-4 carbon
atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8
carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbon
atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7
carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon
atoms, 4-11 carbon atoms, and/or 4-12 carbon atoms, etc., as
appropriate).
In any of the embodiments above, the phrase "salt" or
"pharmaceutically acceptable salt" is intended to include nontoxic
salts synthesized from the parent compound which contains a basic
or acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two. For example, an inorganic acid (e.g., hydrochloric acid,
sulfuric acid, phosphoric acid, or hydrobromic acid), an organic
acid (e.g., oxalic acid, malonic acid, citric acid, fumaric acid,
lactic acid, malic acid, succinic acid, tartaric acid, acetic acid,
trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic
acid, or benzylsulfonic acid), an inorganic base (e.g., sodium
hydroxide, potassium hydroxide, calcium hydroxide, magnesium
hydroxide, or ammonium hydroxide), an organic base (e.g.,
methylamine, diethylamine, triethylamine, triethanolamine,
ethylenediamine, tris(hydroxymethyl)methylamine, guanidine,
choline, or cinchonine), or an amino acid (e.g., lysine, arginine,
or alanine) can be used. Generally, nonaqueous media such as ether,
ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
Lists of suitable salts are found in Remington's Pharmaceutical
Sciences, 18th ed., Mack Publishing Company, Easton, Pa., 1990, p.
1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). For
example, they can be a salt of an alkali metal (e.g., sodium or
potassium), alkaline earth metal (e.g., calcium), or ammonium of
salt.
The diazeniumdiolated compounds of the invention or a composition
thereof can potentially be administered as a pharmaceutically
acceptable acid-addition, base neutralized or addition salt, formed
by reaction with inorganic acids, such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base, such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases, such as mono-, di-, trialkyl, and
aryl amines and substituted ethanolamines. The conversion to a salt
is accomplished by treatment of the base compound with at least a
stoichiometric amount of an appropriate acid. Typically, the free
base is dissolved in an inert organic solvent such as diethyl
ether, ethyl acetate, chloroform, ethanol, methanol, and the like,
and the acid is added in a similar solvent. The mixture is
maintained at a suitable temperature (e.g., between 0.degree. C.
and 50.degree. C.). The resulting salt precipitates spontaneously
or can be brought out of solution with a less polar solvent.
Nitric oxide release from the hybrid diazeniumdiolated compounds
described herein can be determined/detected using known techniques
such as those described in U.S. Pat. Nos. 6,511,991 and 6,379,660;
Keefer, et al., "NONOates(1-Substituted Diazen-1-ium-1,2 diolates)
as Nitric Oxide Donors: Convenient Nitric Oxide Dosage Forms,"
Methods in Enzymology, 28: 281-293 (1996); Horstmann et al.,
"Release of nitric oxide from novel diazeniumdiolates monitored by
laser magnetic resonance spectroscopy," Nitric Oxide, 6(2): 135-41
(2002); and Kitamura et al., "In vivo nitric oxide measurements
using a microcoaxial electrode," Methods Mol. Biol., 279: 35-44
(2004), which are incorporated herein by reference. In general, the
amount of NO produced can be detected by a chemiluminescence
method, electrochemical method, and/or an absorbance method. In
addition, nitric oxide assay kits are commercially available.
The invention provides a pharmaceutical composition comprising the
compound of formula (I) or (II) or a pharmaceutically acceptable
salt thereof and a pharmaceutically acceptable carrier.
In the pharmaceutical compositions described herein, any suitable
pharmaceutically acceptable carrier can be used, and such carriers
are well known in the art. The choice of carrier will be
determined, in part, by the particular site to which the
pharmaceutical composition is to be administered and the particular
method used to administer the pharmaceutical composition.
Suitable formulations include aqueous and non-aqueous solutions,
isotonic sterile solutions, which can contain anti-oxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood or other bodily fluid of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. In one embodiment, the
pharmaceutically acceptable carrier is a liquid that contains a
buffer and a salt. The formulation can be presented in unit-dose or
multi-dose sealed containers, such as ampoules and vials, and can
be stored in a freeze-dried (lyophilized) condition requiring only
the addition of the sterile liquid carrier, for example, water,
immediately prior to use. Extemporaneous solutions and suspensions
can be prepared from sterile powders, granules, and tablets. In one
embodiment, the pharmaceutically acceptable carrier is a buffered
saline solution.
Further carriers include sustained-release preparations, such as
semipermeable matrices of solid hydrophobic polymers containing the
active agent, which matrices are in the form of shaped articles
(e.g., films, liposomes, or microparticles).
The pharmaceutical composition can include carriers, thickeners,
diluents, buffers, preservatives, surface active agents and the
like. The pharmaceutical compositions can also include one or more
additional active ingredients, such as antimicrobial agents,
anti-inflammatory agents, anesthetics, and the like.
The pharmaceutical composition comprising the compound of formula
(I) or (II) or a pharmaceutically acceptable salt thereof can be
formulated for any suitable route of administration, depending on
whether local or systemic treatment is desired, and on the area to
be treated. The pharmaceutical composition can be formulated for
parenteral administration, such as intravenous, intraperitoneal,
intramuscular, or intratumoral injection. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for suspension in liquid prior to
injection, or as emulsions. Additionally, parental administration
can involve the preparation of a slow-release or sustained-release
system, such that a constant dosage is maintained. Preparations for
parenteral administration include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil, and injectable organic esters, such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
also can be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
Desirably, the pharmaceutical composition also can be administered
orally. Oral compositions can be in the form of powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids, or binders may be desirable.
Suitable carriers and their formulations are further described in
A. R. Gennaro, ed., Remington: The Science and Practice of Pharmacy
(19th ed.), Mack Publishing Company, Easton, Pa. (1995).
The compound or a pharmaceutical composition comprising at least
one compound of formula (I) or a pharmaceutically acceptable salt
thereof can be administered in any suitable manner depending on
whether local or systemic treatment is desired, and on the area to
be treated. Desirably, the pharmaceutical composition is
administered orally, but can be administered parenterally, most
preferably by intravenous, intraperitoneal, intramuscular, or
intratumoral injection. By the term "injecting," it is meant that
the pharmaceutical composition is forcefully introduced into the
target tissue. Although more than one route can be used to
administer the pharmaceutical composition, a particular route can
provide a more immediate and more effective reaction than another
route. For regional delivery, the pharmaceutical composition can be
administered intraarterially or intravenously, e.g., via the
hepatic artery for delivery to the liver or the carotid artery for
delivery to the brain.
The compound or a pharmaceutical composition comprising at least
one compound of formula (I) or (II) or a pharmaceutically
acceptable salt thereof can be administered in or on a device that
allows controlled or sustained release of the compound of formula
(I) or (II) or a pharmaceutically acceptable salt thereof, such as
a sponge, biocompatible meshwork, mechanical reservoir, or
mechanical implant. Implants (see, e.g., U.S. Pat. No. 5,443,505),
devices (see, e.g., U.S. Pat. No. 4,863,457), such as an
implantable device, e.g., a mechanical reservoir or an implant or a
device comprised of a polymeric composition, are particularly
useful for administration of the active agents. The pharmaceutical
compositions of the inventive method also can be administered in
the form of sustained-release formulations (see, e.g., U.S. Pat.
No. 5,378,475) comprising, for example, gel foam, hyaluronic acid,
gelatin, chondroitin sulfate, a polyphosphoester, such as
bis-2-hydroxyethyl-terephthalate (BHET), and/or a
polylactic-glycolic acid. Of course, administration of the compound
or pharmaceutical composition can be accomplished via any route
that efficiently delivers the active agents to the target
tissue.
The inventive methods comprise administering an effective amount of
a compound of formula (I) or (II) or a pharmaceutically acceptable
salt thereof. An "effective amount" means an amount sufficient to
show a meaningful benefit in an individual, e.g., promoting at
least one aspect of tumor cell cytotoxicity, or treatment, healing,
prevention, delay of onset, halting, or amelioration of other
relevant medical condition(s) associated with a particular cancer.
Preferably, one or more symptoms of the cancer are prevented,
reduced, halted, or eliminated subsequent to administration of a
compound of formula (I) or (II) or a pharmaceutically acceptable
salt thereof, thereby effectively treating the cancer to at least
some degree.
Effective amounts may vary depending upon the biological effect
desired in the individual, condition to be treated, and/or the
specific characteristics of the compound of formula (I) or (II) or
a pharmaceutically acceptable salt thereof, and the individual. In
this respect, any suitable dose of the compound of formula (I) or
(II) or a pharmaceutically acceptable salt thereof can be
administered to the patient (e.g., human), according to the type of
cancer to be treated. Various general considerations taken into
account in determining the "effective amount" are known to those of
skill in the art and are described, e.g., in Gilman et al., eds.,
Goodman And Gilman's: The Pharmacological Bases of Therapeutics,
8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical
Sciences, 17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of
which is herein incorporated by reference. The dose of the compound
of formula (I) or (II) or a pharmaceutically acceptable salt
thereof desirably comprises about 0.1 mg per kilogram (kg) of the
body weight of the mammal (mg/kg) to about 400 mg/kg (e.g., about
0.75 mg/kg, about 5 mg/kg, about 30 mg/kg, about 75 mg/kg, about
100 mg/kg, about 200 mg/kg, or about 300 mg/kg). In another
embodiment, the dose of the compound of formula (I) or (II)
comprises about 0.5 mg/kg to about 300 mg/kg (e.g., about 0.75
mg/kg, about 5 mg/kg, about 50 mg/kg, about 100 mg/kg, or about 200
mg/kg), about 10 mg/kg to about 200 mg/kg (e.g., about 25 mg/kg,
about 75 mg/kg, or about 150 mg/kg), or about 50 mg/kg to about 100
mg/kg (e.g., about 60 mg/kg, about 70 mg/kg, or about 90
mg/kg).
Reactive Oxygen Species (ROS) are derived from the metabolic
reduction of molecular oxygen. ROS include the superoxide anion
radical (O.sub.2.sup.-), singlet oxygen (.sup.1O.sub.2), hydrogen
peroxide (H.sub.2O.sub.2), and the highly reactive hydroxy radical
(.sup.-OH). These species are highly toxic. ROS normally exist in
all aerobic cells in balance with biochemical antioxidants.
However, oxidative stress disrupts the critical balance because of
excess ROS and/or antioxidant depletion. ROS can cause tissue
damage by reacting with lipids in cellular membranes, nucleotides
in DNA, sulfhydryl groups in proteins, and
crosslinking/fragmentation of ribonucleoproteins. Damage to DNA by
ROS is a major cause of cancer. ROS can damage DNA and the division
of cells with unpaired or misrepaired damage leads to mutations.
The majority of mutations induced by ROS appear to involve
modification of guanine, causing G.fwdarw.T transversions. If it
relates to critical genes such as oncogenes or tumor suppressor
genes, initiation/progression can result. ROS can act at several
steps in a multistate carcinogenesis. Cells characterized by
increased ROS levels often have depressed levels of antioxidant
enzymes.
ROS are also generated when cancer patients are treated with
certain chemotherapeutic agents. For example, ROS generation and
mitochondrial dysfunction are thought to be involved in the
apoptotic response of human H460 NSCLC cancer cells when treated
with a proteasome inhibitor, bortezomib.
A major product of ROS attack in genomic DNA is the premutagenic
lesion 7,8-dihydro-8-oxoguanine (8-oxoG), which causes G-to-T
transversions. The main defense against the 8-oxoG is the base
excision repair (BER) pathway, which in eukaryotes is initiated by
the OGG1 protein, a DNA glycosylase that catalyzes the excision of
8-oxodG from DNA. OGG1 is responsible for over 95% of BER activity
in mammalian cells. A correlation between OGG1 protein expression
levels and IC.sub.50 values for the compound of formula (I) has
been surprisingly discovered. In particular, the compound of
formula (I) is less toxic in the cell lines expressing high levels
of OGG1 protein. This establishes OGG1 as a potential marker for
sensitivity. As a result, in the inventive methods the cancer cell
can have an 8-oxo-dG DNA glycosylase (OGG1) content less than about
25 units (e.g., less than about 20 units, less than about 15 units,
less than about 10 units, or less than about 5 units) relative to
the OGG1 content of the nonmalignant lung epithelial HPL1D which is
100 units.
The amount of OGG1 in a particular cancer cell can be determined by
assays known in the art using, for example, an enzyme-linked
immunosorbent assay (ELISA), real-time PCR (RT-PCR), and/or Western
blot analysis. For example, commercially available kits can be used
to determine the amount of OGG1 in a cell (e.g., an OGG1 assay
kit).
It has been discovered that the PARP-inhibitor/NO-donor dual
prodrugs of formula (I), formula (II), or a pharmaceutically
acceptable salt thereof can effectively kill NSCLC cells, as
evaluated by inhibition of cell proliferation, modulation of DNA
damage/repair, and apoptosis. Therefore, it is envisioned that the
compounds described herein have clinical applications as "stand
alone" therapeutics against cancer cells, particularly cancer cells
characterized by high endogenous levels of ROS and/or low levels of
DNA repair protein OGG1. The inventive compounds described herein
also can serve as adjunct chemosensitizing agents in a wide variety
of cancers.
Cancers treatable with the methods described herein include tumors
associated with the oral cavity (e.g., the tongue and tissues of
the mouth) and pharynx, the digestive system (e.g., the esophagus,
stomach, small intestine, colon, rectum, anus, liver, gall bladder,
and pancreas), the respiratory system (e.g., the larynx, lung, and
bronchus), bones and joints (e.g., bony metastases), soft tissue,
the skin (e.g., melanoma and squamous cell carcinoma), breast, the
genital system (e.g., the uterine cervix, uterine corpus, ovary,
vulva, vagina, prostate, testis, and penis), the urinary system
(e.g., the urinary bladder, kidney, renal pelvis, and ureter), the
eye and orbit, the brain and nervous system (e.g., glioma), and the
endocrine system (e.g., thyroid). The target tissue also can be
located in lymphatic or hematopoietic tissues. For example, the
tumor can be associated with lymphoma (e.g., Hodgkin's disease and
Non-Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute
lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid
leukemia, chronic myeloid leukemia, and the like). The tumor to be
treated is not necessarily the primary tumor. Indeed, the tumor can
be a metastasis of a primary tumor located in a different tissue or
organ.
Specific examples of cancers treatable with the present methods
include, without limitation, acute lymphoblastic leukemia, acute
myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma,
AIDS-related malignancies, anal cancer, cerebellar astrocytoma,
extrahepatic bile duct cancer, bladder cancer,
osteosarcoma/malignant fibrous histiocytoma, brain stem glioma,
ependymoma, visual pathway and hypothalamic gliomas, breast cancer,
bronchial adenomas/carcinoids, carcinoid tumors, gastrointestinal
carcinoid tumors, carcinoma, adrenocortical, islet cell carcinoma,
primary central nervous system lymphoma, cerebellar astrocytoma,
cervical cancer, chronic lymphocytic leukemia, chronic myelogenous
leukemia, clear cell sarcoma of tendon sheaths, colon cancer,
colorectal cancer, cutaneous t-cell lymphoma, endometrial cancer,
ependymoma, esophageal cancer, Ewing's sarcoma/family of tumors,
extracranial germ cell tumors, extragonadal germ cell tumors,
extrahepatic bile duct cancer, eye cancers, including intraocular
melanoma, and retinoblastoma, gallbladder cancer, gastrointestinal
carcinoid tumor, ovarian germ cell tumor, gestational trophoblastic
tumor, hairy cell leukemia, head and neck cancer, Hodgkin's
disease, hypopharyngeal cancer, hypothalamic and visual pathway
glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer,
acute lymphoblastic leukemia, acute myeloid leukemia, chronic
lymphocytic, leukemia, chronic myelogenous leukemia, liver cancer,
non-small cell lung cancer, small cell lung cancer, Hodgkin's
disease, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia,
malignant mesothelioma, malignant thymoma, medulloblastoma,
melanoma, intraocular melanoma, merkel cell carcinoma, metastatic
squamous neck cancer with occult primary, multiple endocrine
neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis
fungoides, myelodysplastic syndrome, chronic myelogenous leukemia,
myeloid leukemia, multiple myeloma, myeloproliferative disorders,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oral cancer, oral cavity and lip cancer,
oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma
of bone, ovarian cancer, ovarian low malignant potential tumor,
pancreatic cancer, paranasal sinus and nasal cavity cancer,
parathyroid cancer, penile cancer, pheochromocytoma, pituitary
tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer,
renal cell (kidney) cancer, transitional cell cancer (e.g. renal
pelvis and ureter), retinoblastoma, rhabdomyosarcoma, salivary
gland cancer, malignant fibrous histiocytoma of bone, soft tissue
sarcoma, sezary syndrome, skin cancer, small intestine cancer,
stomach (gastric) cancer, supratentorial primitive neuroectodermal
and pineal tumors, cutaneous T-cell lymphoma, testicular cancer,
malignant thymoma, thyroid cancer, gestational trophoblastic tumor,
urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
and Wilms' tumor.
The cancers that will be treatable by the methods of the present
invention include, without limitation, brain cancer, bone cancer, a
leukemia, a lymphoma, epithelial cell-derived neoplasia (epithelial
carcinoma) such as basal cell carcinoma, adenocarcinoma,
gastrointestinal cancer such as lip cancer, mouth cancer,
esophageal cancer, small bowel cancer and stomach cancer, colon
cancer, liver cancer, bladder cancer, pancreas cancer, ovary
cancer, cervical cancer, lung cancer, breast cancer and skin
cancer, such as squamous cell and basal cell cancers, prostate
cancer, renal cell carcinoma, and other known cancers that effect
epithelial cells throughout the body.
In an embodiment of the methods of the invention, the cancer is
leukemia, melanoma, lung cancer, colon cancer, brain cancer,
ovarian cancer, breast cancer, prostate cancer, or renal cancer.
Preferably, the cancer is non-small cell lung cancer, such as cells
having one or more characteristics of H1703, H1734, H1693, H1568,
H1373, H2030, H2023, and H1944 cells. In an embodiment, the NSCLC
cell can have one or more of the following characteristics:
TABLE-US-00001 Cell line ROS OGG1 H1703 22.0 14 H1734 18.4 103
H1693 15.1 0.5
Preferably, the NSCLC cell has the characteristics of an H1703 or
H1693 cell line. These NSCLC cell lines can be distinguished from
other lung cancer cell lines, which have one or more biomarkers
outside of the desirable range. For example:
TABLE-US-00002 Cell line ROS OGG1 H441 14.7 28 A549 2.3 260 H1395
8.2 106 H838 6.9 204
Differential NSCLC cells' responsiveness to the drug is believed to
be related to the cancer cells' endogenous level of reactive oxygen
species (ROS). The level of endogenous ROS correlates significantly
with the drug toxicity measured as IC.sub.50 values. Therefore, it
is envisioned that a compound of formula (I) or (II) or a
pharmaceutically acceptable salt thereof shows a synergistic effect
with therapeutics acting through generation of ROS.
In certain embodiments of the invention, the compound of formula
(I) or (II) or a pharmaceutically acceptable salt thereof can be
co-administered with a chemotherapeutic agent. In an embodiment,
the chemotherapeutic agent produces reactive oxygen species (ROS)
in the cancer cell. In this regard, the present invention is
directed a pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a combination of the compound of formula (I)
or (II) or a pharmaceutically acceptable salt thereof and a
chemotherapeutic agent. The cancer cell is the same as described
herein.
Examples of chemotherapeutic agents, including agents that may
produce ROS, include platinum compounds (e.g., cisplatin,
carboplatin, oxaliplatin), alkylating agents (e.g.,
cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard,
thiotepa, melphalan, busulfan, procarbazine, streptozocin,
temozolomide, dacarbazine, bendamustine), antitumor antibiotics
(e.g., daunorubicin, doxorubicin, idarubicin, epirubicin,
mitoxantrone, bleomycin, mytomycin C, plicamycin, dactinomycin),
taxanes (e.g., paclitaxel and docetaxel), antimetabolites (e.g.,
5-fluorouracil, cytarabine, premetrexed, thioguanine, floxuridine,
capecitabine, and methotrexate), nucleoside analogues (e.g.,
fludarabine, clofarabine, cladribine, pentostatin, nelarabine),
topoisomerase inhibitors (e.g., topotecan and irinotecan),
hypomethylating agents (e.g., azacitidine and decitabine),
proteosome inhibitors (e.g., bortezomib), epipodophyllotoxins
(e.g., etoposide and teniposide), DNA synthesis inhibitors (e.g.,
hydroxyurea), vinca alkaloids (e.g., vicristine, vindesine,
vinorelbine, and vinblastine), tyrosine kinase inhibitors (e.g.,
imatinib, dasatinib, nilotinib, sorafenib, sunitinib), monoclonal
antibodies (e.g., rituximab, cetuximab, panetumumab, tositumomab,
trastuzumab, alemtuzumab, gemtuzumab ozogamicin, bevacizumab),
nitrosoureas (e.g., carmustine, fotemustine, and lomustine),
enzymes (e.g., L-Asparaginase), biological agents (e.g.,
interferons and interleukins), hexamethylmelamine, mitotane,
angiogenesis inhibitors (e.g., thalidomide, lenalidomide), steroids
(e.g., prednisone, dexamethasone, and prednisolone), hormonal
agents (e.g., tamoxifen, raloxifene, leuprolide, bicaluatmide,
granisetron, flutamide), aromatase inhibitors (e.g., letrozole and
anastrozole), arsenic trioxide, tretinoin, nonselective
cyclooxygenase inhibitors (e.g., nonsteroidal anti-inflammatory
agents, salicylates, aspirin, piroxicam, ibuprofen, indomethacin,
naprosyn, diclofenac, tolmetin, ketoprofen, nabumetone, oxaprozin),
selective cyclooxygenase-2 (COX-2) inhibitors, or any combination
thereof.
In an embodiment, the chemotherapeutic agent that produces ROS is
an antitumor antibiotic or a proteosome inhibitor, e.g.,
doxorubicin or bortezomib.
Alternatively, the compound of formula (I) or (II) or a
pharmaceutically acceptable salt thereof can be co-administered
with a high energy radiation that produces ROS.
For purposes of the present invention, the term "patient"
preferably is directed to a mammal. Mammals include, but are not
limited to, the order Rodentia, such as mice, and the order
Logomorpha, such as rabbits. It is preferred that the mammals are
from the order Carnivora, including Felines (cats) and Canines
(dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the
order Perssodactyla, including Equines (horses). It is most
preferred that the mammals are of the order Primates, Ceboids, or
Simioids (monkeys) or of the order Anthropoids (humans and apes).
An especially preferred mammal is the human.
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
EXAMPLES
Starting materials were purchased from Aldrich Chemical Co.
(Milwaukee, Wis.) unless otherwise indicated. NMR spectra were
recorded on a Varian UNITY INOVA.TM. spectrometer; chemical shifts
(.delta.) are reported in parts per million (ppm) downfield from
tetramethylsilane. Ultraviolet (UV) spectra were recorded on an
Agilent Model 8453 or a Hewlett-Packard model 8451A diode array
spectrophotometer. Elemental analyses were performed by Midwest
Microlab (Indianapolis, Ind.). Nitric oxide measurements were
performed using a SIEVERS.TM. Nitric Oxide Analyzer (NOA), model
280i (Instruments Business Group, Boulder, Colo.). Chromatography
was performed on a Biotage SP1.TM. FLASH.TM. Purification System.
Prepacked silica gel flash chromatography columns were purchased
from Silicycle (Quebec City, Canada) or from Yanazen Science Inc.
(San Bruno, Calif.).
Example 1
This example demonstrates the synthesis of 2,4-dinitrophenyl
4-[5-[(4-oxo-3H-phthalazin-1-yl)methyl]-benzoyl]piperazine-1-yl-1-ium-1,2-
-diolate (JS-59-183) in an embodiment of the invention. See FIG.
2.
3-[(4-Oxo-3H-phthalazin-1-yl)methyl]benzoic acid, 1a, was prepared
as described by Menear et al. (J. Med. Chem., 51: 6581-6591
(2008)). To a slurry of 557 mg (1.6 mmol) of the hydrochloride salt
of O.sup.2-(2,4-dinitrophenyl)
1-(piperazine-1-yl)diazene-1-ium-1,2-diolate, 2 (Shami et al., J.
Med. Chem., 49: 4356-4366 (2006)), 448 mg (1.6 mmol) of 1a and 608
mg (1.6 mmol) of
((2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) (HATU) in 5 mL of dimethylacetamide was added
1.02 mL (6 mmol) of diisopropyl ethylamine. The resulting solution
was stirred at room temperature for 1 h, followed by the addition
of 40 mL of water. The resulting precipitate was collected by
filtration and recrystallized from ethanol giving 827 mg of an
off-white solid: uv (acetonitrile), .lamda..sub.max 294 nm
(.epsilon.=18.9 mM.sup.-1 cm.sup.-1); mp 114-17.degree. C.; .sup.1H
NMR (DMSO-d.sub.6), .delta. 3.63-3.68 (m, 8H), 4.38 (s, 2H),
7.28-7.30 (m, 1H), 7.39-7.46 (m, 3H), 7.81-7.98 (m, 4H), 8.26 (d,
1H) J=7.42 Hz, 8.56-8.59 (dd, 1H) J=2.7, 7.42 Hz, 8.88 (d, 1H)
J=2.7 Hz, 12.06 (s, 1H).
Example 2
This example demonstrates the synthesis of 2,4-dinitrophenyl
4-[2-fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]-benzoyl]piperazine-1-yl--
1-ium-1,2-diolate (JS-60-178) in an embodiment of the invention.
See FIG. 2.
To a mixture of 167 mg (0.560 mmol) of
2-fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]benzoic acid, 1b, 209
mg (0.6 mmol) of the hydrochloride salt of
O.sup.2-(2,4-dinitrophenyl)
1-(piperazine-1-yl)diazen-1-ium-1,2-diolate, 2 (Shami et al., J.
Med. Chem., 49: 4356-4366 (2006)), and 247 mg of HATU, were added
0.247 mL (1.8 mmol) of triethylamine and 5 mL of dimethylformamide;
the resulting solution was stirred overnight. Water (5 mL) was
added to the reaction mixture, which was stirred at room
temperature for 1 h. The resulting yellow precipitate was collected
by filtration and the solid was washed with ice-cold 1:1 water:DMF,
then allowed to dry to give 264 mg of a yellow solid. The material
was recrystallized from methanol:dichloromethane:ether: uv,
(acetonitrile), .lamda..sub.max 295 nm (.epsilon.=20.5 mM.sup.-1
cm.sup.-1); mp 150-3, .sup.1H NMR (DMSO-d.sub.6), .delta. 3.41 (b,
1H), 3.58 (b, 1H), 3.72 (b, 1H), 3.84 (b, 1H), 4.33 (s, 2H), 7.25
(t, 1H) J=9 Hz, 7.37-7.46 (m, 2H), 7.82 (t, 2H) J=9 Hz, 7.87-7.97
(m, 3H), 8.24 (d, 1H) J=7.8 Hz, 8.54-8.57 (dd, 1H) J=2.4, 7.8 Hz,
8.86 9 (d, 1H) J=2.4 Hz, 12.58 (s, 1H).
Example 3
This example demonstrates the synthesis of 2,4-dinitrophenyl
4-[2-benzyloxy benzamide]piperazine-1-yl-1-ium-1,2-diolate
(JS-59-13) in an embodiment of the invention. See FIG. 3.
To a slurry of 245 mg (0.904 mmol) of
2-[(1-carboxy)benzyloxy]benzamide, 3, prepared as described by
Menear et al. (Bioorg. Med. Chem. Lett., 18: 3942-3945 (2008)), in
10 mL of dimethylformamide was added 0.100 mL of thionyl chloride
and stirred at room temperature for 30 min. To the reaction mixture
were added 313 mg (0.9 mmol) of the hydrochloride salt of 2 and 0.5
mL of triethylamine. After stirring overnight, water was added. The
solid product was extracted with dichloromethane and washed with
dilute sodium bicarbonate solution, dried over sodium sulfate
filtered through magnesium sulfate and evaporated to give 298 mg of
a solid. The product was chromatographed on silica gel, eluted with
9:1 dichloromethane:methanol: uv, (acetonitrile), .lamda..sub.max
298 nm (.epsilon.=000 mM.sup.-1 cm.sup.-1); mp 110-111.degree. C.;
.sup.1H NMR (DMSO-d.sub.6), .delta. 3.73 (b, 8H), 5.35 (s, 2H),
7.12 (t, 2H) J=7.8 Hz, 7.35 (d, 1H) J=8.4 Hz, 7.44-7.62 (m, 4H),
7.69 (t, 1H) J=8.4 Hz, 7.76 (d, 1H) J=7.8 Hz, 7.94 (d, 1H) J=9 Hz,
8.55 (dd, 1H) J=2.73, 9.0 Hz, 8.80 (d, 1H) J=2.73 Hz; .sup.13C NMR
(DMSO d.sub.6), .delta. 50.29, 70.04, 101.27. 113.98, 116.8, 118.5,
121.88, 127.22, 126.56, 127.31, 129.30, 129.37, 130.13, 134.21,
135.52, 135.78, 136.95, 137.29, 142.62, 153.16, 160.16, 169.26.
Anal., C, H, N: Calcd. for C.sub.25H.sub.27N.sub.7O.sub.9: C,
53.10; H, 4.10; N, 17.34. Found: C, 53.80; H, 4.11; N, 17.47.
Example 4
This example demonstrates the synthesis of
1-[1-(N,N-dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-dinitrophenyl
4-[2-fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]-benzoyl]piperazine-1-yl--
1-ium-1,2-diolate (JS-65-103) in an embodiment of the
invention.
To a solution of 24 mg (0.08 mmol) of
2-fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]benzoic acid (Menear
et al., J. Med. Chem., 51: 6581-6591 (2008)) and 30 mg (0.08 mmol)
of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) (HATU) in 2.5 mL of N,N-dimethylformamide was
added 31 .mu.L (0.18 mmol) of diisopropyl ethylamine (DIPEA). To
the solution was gradually added 36 mg (0.08 mmol) of the
hydrochloride salt in 2.5 mL of N,N-dimethylformamide and the
resulting solution was stirred at room temperature overnight. The
solution was treated with 10 mL of water, and the resulting slurry
was stirred for 1 hr, centrifuged, and the liquid decanted. The
precipitate was washed with water, then extracted with ethyl
acetate and washed with 5% sodium bicarbonate. The organic layer
was dried over sodium sulfate, filtered through a layer of
magnesium sulfate, and concentrated under vacuum to give 31 mg of a
beige solid (JS-65-103): uv (0.2% DMSO/ethanol), .lamda..sub.max
287 nm (.epsilon.=32 mM.sup.-1 cm.sup.-1); .sup.1H NMR
(acetone-d.sub.6), .delta. 3.30 (s, 6H), 3.58-3.97 (m, 8H),
7.14-7.22 (m, 1H), 7.44-7.54 (m, 2H), 7.79 (s, 1H), 7.80-7.88 (m,
1H), 7.94-7.96 (m, 2H), 8.32-8.34 (m, 1H), 8.86 (s, 1H), 11.75 (s,
1H); .sup.13C NMR (acetone-d.sub.6) .delta. 37.91, 41.76, 50.93,
51.24, 105.37, 116.64, 116.85, 124.55, 124.73, 126.1, 126.31,
127.31, 129.49, 130.19, 130.45, 132.1, 132.23, 132.34, 132.80,
134.22, 145.74, 154.8, 155.2, 160.44, 165.15.
The material contained impurities from N,N-dimethylformamide,
dichloromethane, and other minor impurities. For further
decomposition and biological screening, a portion of the compound
was purified on a Phenomenex LUNA.TM. C18 column, 3 .mu.m,
150.times.2.0 mm, with a gradient consisting of water and
acetonitrile containing 0.1% formic acid. HRMS (ESI) m/z calculated
for C.sub.28H.sub.27FN.sub.11O.sub.10 [M+H].sup.+696.19209. found
696.19276.
Example 5
This example demonstrates the determination of intracellular
reactive oxygen/nitrogen species and nitric oxide of specific
compounds of formula (I) in an embodiment of the invention.
Intracellular level of reactive oxygen/nitrogen species was
quantified by the oxidation of the ROS/RNS-sensitive fluorophore
5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate
(Invitrogen, Carlsbad, Calif.). Cells growing on six-well plates
(6.times.10.sup.5/well) were loaded with 5 .mu.M
5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate
in Hanks' balanced salt solution (HBSS) at 37.degree. C. and 5%
CO.sub.2. After 30 min of incubation, HBSS containing the probe was
removed, cells were rinsed with HBSS, and 3 ml of fresh HBSS was
added to each well followed by the addition of compounds (10 .mu.M)
or DMSO as a control. After 60 min, the cells were collected by
scraping in HBSS, and 2',7'-dichlorofluorescein (DCF) fluorescence
was measured by using a PerkinElmer Life and Analytical Sciences
(Waltham, Mass.) LS50B luminescence spectrometer with the
excitation source at 488 nm and emission at 530 nm.
The intracellular level of nitric oxide after treatment with the
compounds JS-59-183 or JS-60-178 was quantified by using the
NO-sensitive fluorophore 4-amino-5-methylamino-2,7
difluorofluorescein (DAF-FM) diacetate (Invitrogen, Carlsbad,
Calif.). Cells growing on six-well plates were loaded with 2.5
.mu.M DAF-FM diacetate in HBSS at 37.degree. C. and 5% CO.sub.2.
After 30 min of incubation, the cells were rinsed with HBSS to
remove excess probe, and compounds in fresh HBSS was added to the
cells at 10 .mu.M final concentration. After 30 min incubation, the
fluorescence of the benzotriazole derivative formed on DAF-FM's
reaction with aerobic NO was analyzed by using a PerkinElmer Life
and Analytical Sciences LS50B luminescence spectrometer with the
excitation source at 495 nm and emission at 515 nm. All experiments
were performed at least three times, each time at least in
triplicate.
These assays demonstrate that the PARP-inhibitor/NO-donor dual
prodrugs JS-59-183 and JS-60-178 are cell permeable and decompose
within the cell releasing NO (FIG. 4A), as demonstrated with
NO-specific reagent DAF-FM diacetate. There was also an increase in
ROS generation after treatment with JS-59-183 and JS-60-178, as
detected by the oxidation-sensitive fluorophore DCF (FIG. 4B).
Example 6
This example demonstrates the inhibition of PARP of specific
compounds of formula (I) in an embodiment of the invention.
PARP enzyme inhibition was measured using HT Universal Colorimetric
96-well PARP assay kit (Trevigen, Gaithersburg, Md.), according to
manufacturer's protocol with small modifications. GSH (1 mM final
concentration) was added to the wells containing inhibitors, to
allow activation of the prodrugs. The absorbance at 450 nm was
measured.
The PARP inhibitory activities of JS-59-183 and JS-60-178 were
comparable to that of Olaparib. PARP enzyme IC.sub.50 values
estimated for JS-60-178 were 2.9 nM, for JS-59-183 8.25 nM, and for
Olaparib 2.0 nM (FIG. 5).
Example 7
This example demonstrates the inhibition of cell proliferation of
specific compounds of formula (I) in an embodiment of the
invention.
Cell lines were obtained from the American Type Culture Collection
(Manassas, Va.) and cultured according to the supplier's protocol.
For proliferation assays cells were seeded at 1.times.10.sup.4 per
well (H1693, H322M, H1703, H1944, H1355, H2122, H441, H1568) or
5.times.10.sup.3 per well (H460, H1792, A549, H2023, H2030, H23) in
96-well plates and allowed to adhere for 24 h. Compounds were
prepared as 10 mM stock solutions in DMSO. Increasing drug
concentrations in 10 .mu.l of PBS were added to 100 .mu.l of the
culture medium for 72 h. MTT assay (Promega, Madison, Wis.) was
performed according to the manufacturer's protocol. Each
concentration was represented in six repeats, and the screening was
performed as at least two independent experiments. IC.sub.50 values
were calculated by using Sigma Plot software (Systat Software,
Inc., San Jose, Calif.).
JS-59-183 and JS-60-178 inhibited growth of NSCLC cell lines with
IC.sub.50 concentrations ranging from 3 to 20 .mu.M. Both IC.sub.50
and total growth inhibition (TGI) values for JS-59-183 and
JS-60-178 were lower than those of Olaparib for the most sensitive
cell lines, suggesting that NO released from the prodrug upon
activation with GSH contributes to the cytotoxicity. See Table
1.
TABLE-US-00003 TABLE 1 JS-60-178 JS-59-183 Olaparib Cell line
IC.sub.50 [.mu.M] TGI [.mu.M] IC.sub.50 [.mu.M] TGI [.mu.M]
IC.sub.50 [.mu.M] TGI [.mu.M] H1568 3.0 4.0 6.5 9.0 36 113 H1703
4.3 6.7 6.8 11.2 20 60 H441 4.5 6.2 8.1 11.7 28 84 H1693 5.4 6.6
5.3 7.3 19 65 H2122 7.5 11.0 11.2 25.0 38 140 H322M 7.9 11.6 9.0
14.0 50 360 H1355 8.2 15.0 11.4 23.0 33 113 H23 8.4 12.3 7.5 15.2
10 70 H2030 9.2 15.2 13.8 25.0 34 117 H1944 10.5 19.0 14.0 28.0 25
88 H1792 12.7 19.0 14.0 20.0 16 38 A549 13.5 22.0 16.8 26.0 28 100
H2023 15.0 25.0 19.4 32.5 20 85 H460 16.4 24.0 13.8 24.8 12 37
Toxicities of JS-59-183 and JS-60-178 (expressed as IC.sub.50 and
TGI concentrations) correlated with the preexisting endogenous
level of ROS in NSCLC cells (FIG. 6). Other related factors,
including levels of 8-oxoguanine DNA glycosylase (OGG1) and
peroxide scavenging enzyme peroxiredoxin 1 (PRX1), also correlated
with IC.sub.50/TGI values for JS-59-183 and JS-60-178. As shown in
Table 2, the toxicities of JS-60-178 and JS-59-183 but not olaparib
correlated with endogenous levels of ROS, PRX1 and OGG1
(P.ltoreq.0.05 considered significant, by Pearson linear
correlation or Spearman regression analysis, when appropriate).
TABLE-US-00004 TABLE 2 ROS PRX1 OGG1 JS-60-178 IC.sub.50 0.002
0.029 0.006 TGI 0.001 0.010 0.002 JS-59-183 IC.sub.50 0.003 0.017
0.002 TGI 0.002 0.004 0.001 olaparib IC.sub.50 NS (0.620) NS
(0.539) NS (0.336) TGI NS (0.530) NS (0.546) NS (0.350)
The alkaline comet assay was performed as described (Romanowska et
al., Free Radical Biol. Med., 43: 1145-1155 (2007)). Western blot
analysis was performed as described previously (Maciag et al., J.
Pharmacol. Exp. Ther., 336: 313-320 (2011)). Primary antibodies for
cleaved caspase 7 (Cell Signaling Technology, Danvers, Mass.) were
used.
Twenty-four hours treatment with JS-60-178 resulted in significant
DNA strand break damage as evidenced by Comet assay. More
specifically, treatment with JS-60-178 at 5 .mu.M concentration
resulted in a stronger comet signal, compared with much higher
concentrations of Olaparib (20 .mu.m). In addition, a strong
apoptotic signal was observed for JS-60-178 (10 .mu.m) and JS-59-83
(10 .mu.m) (as evidenced by cleaved caspase 7), while the same
concentration of Olaparib (10 .mu.m) did not trigger apoptosis.
DMSO alone was used as a control.
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. The terms "comprising," "having,"
"including," and "containing" are to be construed as open-ended
terms (i.e., meaning "including, but not limited to,") unless
otherwise noted. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *